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. 2014 Apr 24;176(3):310–319. doi: 10.1111/cei.12294

Adjuvant effects of aluminium hydroxide-adsorbed allergens and allergoids – differences in vivo and in vitro

B Heydenreich *, I Bellinghausen *,✉, L Lund †, H Henmar †, G Lund †, P Adler Würtzen †, J Saloga *
PMCID: PMC4008974  PMID: 24528247

Abstract

Allergen-specific immunotherapy (SIT) is a clinically effective therapy for immunoglobulin (Ig)E-mediated allergic diseases. To reduce the risk of IgE-mediated side effects, chemically modified allergoids have been introduced. Furthermore, adsorbance of allergens to aluminium hydroxide (alum) is widely used to enhance the immune response. The mechanisms behind the adjuvant effect of alum are still not completely understood. In the present study we analysed the effects of alum-adsorbed allergens and allergoids on their immunogenicity in vitro and in vivo and their ability to activate basophils of allergic donors. Human monocyte derived dendritic cells (DC) were incubated with native Phleum pratense or Betula verrucosa allergen extract or formaldehyde-or glutaraldehyde-modified allergoids, adsorbed or unadsorbed to alum. After maturation, DC were co-cultivated with autologous CD4+ T cells. Allergenicity was tested by leukotriene and histamine release of human basophils. Finally, in-vivo immunogenicity was analysed by IgG production of immunized mice. T cell proliferation as well as interleukin (IL)-4, IL-13, IL-10 and interferon (IFN)-γ production were strongly decreased using glutaraldehyde-modified allergoids, but did not differ between alum-adsorbed allergens or allergoids and the corresponding unadsorbed preparations. Glutaraldehyde modification also led to a decreased leukotriene and histamine release compared to native allergens, being further decreased by adsorption to alum. In vivo, immunogenicity was reduced for allergoids which could be partly restored by adsorption to alum. Our results suggest that adsorption of native allergens or modified allergoids to alum had no consistent adjuvant effect but led to a reduced allergenicity in vitro, while we observed an adjuvant effect regarding IgG production in vivo.

Keywords: allergen, allergoid, aluminium hydroxide, dendritic cells, Th1/Th2

Introduction

The prevalence of allergic diseases such as allergic rhinitis or allergic asthma has increased in the last decades. Atopic diseases are characterized by an imbalance of the T cell response [dominance of T helper type 2 (Th2) versus regulatory T cells (Treg)] against otherwise harmless antigens such as grass or tree pollen [1]. Allergen-specific immunotherapy (SIT) is the only disease-modifying treatment known so far [2]. During SIT increasing amounts of specific allergens are administered to achieve immunological tolerance and to prevent a progression of the disease, e.g. from allergic rhinitis to asthma [3]. Successful SIT is characterized by a shift towards a Th1-dominated immune response accompanied by the production of the cytokines interleukin (IL)-10 and transforming growth factor (TGF)-β by induced regulatory T cells (iTreg) and the induction of allergen-specific non-immunoglobulin (Ig)E antibodies such as IgG4 [4–7].

To reduce IgE-mediated side effects occurring during SIT, Marsh et al. introduced chemically modified allergens, so-called allergoids [8]. Modification with aldehydes results in the disruption of the three-dimensional structure of the proteins due to the reactivity with primary amines of the polypeptide chains leading to intra-and intermolecular bonds. Ideally, three-dimensional B cell epitopes should be destroyed during this process, while linear T cell epitopes remain intact to ensure immunogenicity [9–12].

Aluminium hydroxide or aluminium phosphate, commonly referred to as alum, are widely used as adjuvants for vaccines as well as for SIT to enhance the immune response [13,14]. The mechanisms underlying the adjuvant effect are still not fully understood. Different suggestions exist to explain the immunostimulatory effect of alum. One explanation is that alum might exert a depot effect by which the antigen is released slowly and the presentation to antigen-presenting cells is prolonged [15,16]. Recently, the involvement of the protein complex Nalp3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome has been discussed to play a role in the adjuvant effect of alum. Several studies have shown that alum stimulates the release of proinflammatory cytokines such as IL-1β, IL-18 or IL-33 in vitro by activation of caspase 1, a component of the Nalp3 inflammasome complex [17,18]. However, the direct involvement of the Nalp3 inflammasome with regard to an immunostimulatory effect of alum is not fully clear, and remains controversial. There are studies which exclude a direct action of alum on the Nalp3 inflammasome regarding an adjuvant effect [19,20]. Furthermore, there is a suggestion that alum stimulates the production of prostaglandin E2 (PGE2) in macrophages. Thereupon, PGE2 will regulate the Th2 immune response in vivo independently of the Nalp3 inflammasome [21].

Our present study aimed to analyse the effect of alum on the stimulation of basophils and the capacity of alum-pulsed dendritic cells (DC) to activate T cells. Therefore we applied alum-adsorbed native allergens and formaldehyde-or glutaraldehyde-modified allergoids and compared them with their corresponding unadsorbed antigens with regard to their allergenicity and immunogenicity in vitro. Furthermore, we investigated antibody responses after immunization of mice with alum-adsorbed and unadsorbed allergens and allergoids in vivo.

Materials and methods

Allergoid preparation/alum adsorption

Native allergen extracts [10 mg/ml in phosphate-buffered saline (PBS)] of Betula verrucosa (Bet v) and Phleum pratense (Phl p) (ALK-Abello, Hørsholm, Denmark) were used for modification with formaldehyde or glutaraldehyde, as described previously [22]. Alum adsorption was performed by dilution of the allergen/allergoids in Coca buffer (0·25% sodium hydrogen carbonate and 0·5% sodium chloride) and slowly adding Alhydrogel® 1·3% [Al(OH)3] (Brenntag Biosector, Fredrikssund, Denmark), as described previously [23].

Mice immunization and analysis of Phl p-specific IgG

All experiments described in this paper were conducted in accordance with Danish legislation. Six to 8-week-old female BALB/c mice, which were bred under a specific pathogen-free environment at Pipeline Biotech A/S (Trige, Denmark), were immunized intraperitoneally (i.p.) with 0·1 ml alum-adsorbed or unadsorbed Phl p extract or Phl p allergoid corresponding to 10 000 SQ-U/dose six times every second week. Blood samples were drawn before immunization and 1 week after the second, third, fifth and sixth immunization. Sera were prepared from each individual blood sample and Phl p-specific IgG antibodies were analysed by direct enzyme-linked immunosorbent assay (ELISA), as described previously [23]. Experiments were performed in accordance with current federal, state and institutional guidelines.

Patients

Buffy coats were obtained from 18 allergic donors sensitized to Phl p or Bet v with an immuno CAP class ≥ 2 (Transfusion Centre, Mainz, Germany). Specific sensitization was verified by detection of allergen-specific IgE in the sera (ImmunoCAP®-specific IgE blood test; Phadia AB, Uppsala, Sweden). The study was approved by the local ethics committee. Informed consent was obtained from all subjects before the study.

Generation of monocyte-derived DC

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll Paque 1077 g/ml (PAA Laboratories GmbH, Cölbe, Germany) density centrifugation from heparinized blood. CD14+ cells were enriched by incubation of 5 × 106 PBMC in a 12-well-plate (Greiner, Frickenhausen, Germany) with 1 ml/well Iscove's modified Dulbecco's medium containing L-glutamine and 25 mM Hepes (IMDM; PAA Laboratories GmbH) and 3% autologous heat-inactivated plasma at 37°C for 45 min. Non-adherent cells were washed twice with prewarmed PBS. The remaining monocytes were incubated with 1·5 ml/well IMDM, 1% autologous plasma, 1000 U/ml IL-4 (Miltenyi Biotec, Bergisch Gladbach, Germany) and 200 U/ml granulocyte–macrophage colony-stimulating factor (GM-CSF, Leukine®; Immunex Corp., Seattle, WA, USA). On day 6 the immature DC were pulsed with native allergen and allergoids or the corresponding alum-adsorbed allergens and allergoids in different concentrations (4 and 20 μg/ml) and were stimulated with 1000 U/ml tumour necrosis factor (TNF)-α, 2000 U/ml IL-1β (Miltenyi Biotec) and 1 μg/ml PGE2 (Cayman Chemical, Ann Arbor, MI, USA). After 48 h the mature DC were harvested, washed twice with PBS, and used for T cell stimulation assays. Mature DC expressed high levels (> 90%) of CD80, CD83, CD86 and major histocompatibility complex (MHC) class II molecules, controlled by flow cytometry.

For analysis of IL-1β expression, immature DC were stimulated with allergen or lipopolysaccharide (LPS) or were left untreated. Additionally, different alum preparations were added in different concentrations (Imject Alum; Pierce, Rockford, IL, USA and Alhydrogel 1·3%; Brenntag Biosector). After 2–48 h of stimulation, supernatants were harvested and the remaining DC were washed twice with PBS and used for total RNA extraction or lysed in 50 ml lysis buffer [50 mM Hepes, pH 7,4, 150 mM NaCl, 1·5 mM MgCl2, 1 mM ethylenediamine tetraacetic acid (EDTA), 10% glycerol, 1% Triton-X-100] on ice for 30 min. Protein content of the lysates was determined using the BCA protein assay (Pierce).

Isolation of CD4+ T cells and co-culture of T cells and autologous native allergen-, allergoid-or alum-adsorbed allergen/allergoid-pulsed DC

Autologous CD4+ T cells were obtained from PBMC using antibody-coated paramagnetic MicroBeads (MACS; Miltenyi Biotec), according to the manufacturer's protocol. Separation was controlled by flow cytometry (purity > 98% CD4+ T cells). For the proliferation assay 1 × 105 CD4+ T cells were co-cultured in triplicate with 1 × 104 DC, pulsed with native allergen or allergoid or the corresponding allergens and allergoids adsorbed to alum, in 200 μl IMDM, 5% autologous plasma. After 5 days, the cells were pulsed with 37 kBq/well of [3H]-TdR ([methyl-3H]-thymidine; ICN, Irvine, CA, USA) for 6 h. [3H]-TdR incorporation was measured in a beta counter (1205 Betaplate; LKB Wallac, Turku, Finland). For the cytokine assay, 5 × 105 CD4+ T cells and 5 × 104 native allergen-, allergoid-or alum-adsorbed allergen/allergoid-pulsed DC were co-cultured in a 48-well plate in 1 ml IMDM, 5% autologous plasma. After 48 h, supernatants were harvested to measure production of IL-2. For all other cytokines T cells were restimulated for 24 h with freshly generated native allergen-, allergoid-or alum-adsorbed allergen/allergoid-pulsed DC on day 7.

Quantification of cytokine production

Human IL-2, IL-4, IL-10, IL-13 (BD Biosciences, San Jose, CA, USA), and IFN-γ (Mabtech AB, Nacka Strand, Sweden) were measured by ELISA according to the instructions of the manufacturers of the employed pairs of antibodies. Additionally, cytokines in the supernatants were analysed by cytometric bead array (CBA), according to the manufacturer's protocol (CBA Human Soluble Protein Master Buffer Kit and Human IL-1β Flex Set, both from BD Biosciences).

Determination of histamine and leukotriene release from activated basophils

Allergenicity was tested by histamine and leukotriene release of activated basophils using the histamine ELISA (IM2015 enzyme immunoassay kit; Immunotech, Marseille, France), as described previously [24], and for leukotriene release the cellular antigen stimulation test (CAST®-2000 ELISA; Bühlmann Laboratories AG, Schönenbuch, Switzerland), according to the manufacturer's recommendations. Briefly, dextran (250 μl) was added to 1 ml EDTA blood and incubated for 90 min at room temperature to allow sedimentation of erythrocytes. Then, peripheral blood leucocytes (PBL) were centrifuged at 130 g for 15 min and resuspended in 1 ml stimulation buffer containing IL-3. PBL (200 μl) were stimulated with 50 μl native allergen, allergoid or alum-adsorbed allergen/allergoid at different concentrations or anti-IgE receptor antibody as positive control for 40 min at 37°C. Finally, the cells were centrifuged and supernatants were stored at −20°C until quantitative analysis of leukotriene concentration by ELISA.

Surface phenotyping by flow cytometric analysis

For analysis of surface marker expression, 5 × 105 T cells or 5 × 104 DC were labelled with specific mouse anti-human monoclonal antibodies (mAbs) for 20 min at 4°C. The following antibodies were used: AlexaFluor 647-conjugated CD4 (MT310; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), phycoerythrin (PE)-conjugated CD80 (L307·4), CD83 (HB15e), CD86 [2331 (FUN-1)], fluorescein isothiocyanate (FITC)-conjugated human leucocyte antigen D-related (HLA-DR) (L243) and mouse IgG isotype controls (all from BD Biosciences). After incubation, the cells were washed and analysed in a fluorescence activated cell sorter (FACS)Calibur (Becton Dickinson, Mountain View, CA, USA) equipped with CellQuest Software.

Determination of cell viability

To analyse apoptotic and necrotic cells, 1 × 105 immature or mature allergen-, allergoid-or alum-adsorbed allergen/allergoid-pulsed DC were incubated with PE-conjugated annexin V and 7-aminoactinomycin D (7-AAD) (both from BD Biosciences) in binding buffer (10 mM HEPES, 140 mM NaCl, 2·5 mM CaCl2) for 15 min in the dark at room temperature (RT). After incubation, 200 μl binding buffer was added and the cells were analysed by flow cytometry. Thereby, early apoptotic annexin V positive cells can be distinguished from necrotic or late apoptotic cells, which are positive for both annexin V and 7-ADD.

RNA isolation and quantitative real-time polymerase chain reaction (PCR)

Total RNA was isolated from immature DC according to the manufacturer's protocol (RNeasy Mini Kit; Qiagen, Hilden, Germany); 150 ng RNA were used for cDNA synthesis (QuantiTect® Reverse Transcription Kit; Qiagen). To analyse gene expression of IL-1β, quantitative real-time PCR was performed according to the manufacturer's protocol using the QuantiFast® SYBR® Green PCR Kit and QuantiTect Primer Assays (Qiagen) for β-actin [Hs_ACTB_2_SG (QT01680476)] and IL-1β [Hs_IL1B_1_SG (QT00021385)].

Statistics

Analysis of variance (anova) was used for analysis of variance between different experimental groups. Student's paired t-test was used to test the statistical significance of the results; P ≤ 0·05 was considered significant.

Results

High alum concentrations induce apoptosis in mature DC

First, we determined the influence of alum on cell viability of alum-treated immature and mature human DC. After incubation with native allergen/allergoids or alum-adsorbed allergens/allergoids, DC were stained with trypan blue to determine dead cells. In all experiments, a higher amount of DC were dead after incubation with alum-adsorbed antigens compared to the unadsorbed antigens. Thereupon, the cell viability was analysed by staining immature and mature DC with annexin V and 7-AAD to discriminate between apoptotic and necrotic cells. Up to 15% of immature and 10% of mature DC were positive for both annexin V and 7-AAD when they were incubated with allergen or allergoid or when they were left untreated (medium control). Incubation with alum-adsorbed allergens and allergoids or with alum alone strongly increased the amount of annexin V+7-AAD+ cells particularly and significantly in mature DC (Fig. 1). For further T cell stimulatory experiments, only viable DC were used.

Figure 1.

Figure 1

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Alum-adsorbed allergens and allergoids induce enhanced apoptosis compared to the corresponding unadsorbed allergens and allergoids mainly in mature dendritic cells (DC). To determine apoptotic and necrotic cells, immature DC were stimulated on day 6 with alum-adsorbed allergens or allergoids or the unadsorbed allergens/allergoids. Additionally, DC were left immature (a) or matured by additional treatment with proinflammatory cytokines (b). After 48 h DC were harvested and stained with annexin V and 7-aminoactinomycin D (7-AAD). M = medium control; TT = tetanus toxoid 1 μg/ml; A = native Phleum (Phl) or Betula (Bet) allergen extract 20 μg/ml; F = formaldehyde-modified allergoid 20 μg/ml; G = glutaraldehyde modified allergoid 20 μg/ml; mDC = mature DC. Shown are the mean ± standard error of the mean (s.e.m.) from two (a) and four (b) allergic donors. *Statistically significant differences (P < 0·01) between alum-adsorbed and unadsorbed allergens/allergoids.

Decreased histamine and leukotriene release by basophils after stimulation with alum-adsorbed allergens and allergoids

To analyse allergenicity, basophils of allergic donors were stimulated with different concentrations of alum-adsorbed allergens and allergoids or the corresponding unadsorbed allergens and allergoids and the subsequent release of the mediator histamine as well as sulphidoleukotriene LTC4, and its metabolites were quantified by ELISA. Generally, the histamine and leukotriene release by basophils showed great donor variability. As expected from previous observations [22], mediator release was decreased strongly after stimulation with glutaraldehyde-modified allergoids compared to its native allergen extracts and formaldehyde-modified allergoids (Fig. 2). Compared to unadsorbed allergens and allergoids, alum-adsorbed antigens led to a further decreased histamine release primarily for the allergoids, whereas leukotriene release by basophils was reduced for all allergen and allergoid preparations (Fig. 2). To exclude a general toxic effect of alum on basophils, cells were stimulated with alum in combination with the anti-IgE receptor antibody as positive control. No significant differences regarding leukotriene release could be observed after stimulation with alum and anti-IgE receptor antibody compared to the antibody alone (data not shown).

Figure 2.

Figure 2

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Histamine and leukotriene release of basophils is decreased after stimulation with alum-adsorbed extracts and with glutaraldehyde-modified allergoids. Basophils of allergic donors were stimulated with different concentrations of alum-adsorbed and unadsorbed native allergens or allergoids. Histamine (a) and leukotriene release (b) were quantified by enzyme-linked immunosorbent assay (ELISA). Phl = native Phleum (Phl) allergen extract; Phl F = Phl formaldehyde-modified allergoid; Phl G = Phl glutaraldehyde modified allergoid. Left panels show one representative single experiment [mean ± standard error of the mean (s.e.m.) from triplicates] and right panels show results from nine (a) and six (b) allergic donors (means ± s.e.m.). Significant differences are found between Phl G alum and Phl alum or Phl G alum and Phl, using non-parametric rank test, Dunn's multiple comparison.

Alum-adsorbed allergens-, allergoids-and their corresponding unadsorbed antigen-pulsed DC possess similar T cell stimulatory capacity in vitro

To assess immunogenicity in vitro, DC were stimulated with two different concentrations of alum-adsorbed allergens or allergoids or the corresponding unadsorbed antigens and were co-cultured with autologous CD4+ T cells. Co-cultures were analysed for T cell proliferation and production of IL-2, IL-4, IL-13, IL-10 and IFN-γ. Allergen-specific T cell proliferation increased depending on allergen or allergoid concentrations. Both alum-adsorbed and unadsorbed glutaraldehyde-modified allergoids showed a significantly decreased proliferation (Fig. 3) and IL-2 production (data not shown) compared to the corresponding allergen extracts and formaldehyde-modified allergoids. Apart from this, the proliferation and IL-2 production did not differ between adsorbed and unadsorbed allergens. Alum had no effect on the T cell stimulatory capacity of native allergens or allergoids in vitro.

Figure 3.

Figure 3

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Alum-adsorbed allergen-or allergoid-pulsed dendritic cells (DC) exhibit similar T cell stimulatory capacity compared to the corresponding unadsorbed allergen-or allergoid-pulsed DC. Alum-adsorbed and unadsorbed allergen-or allergoid-pulsed DC were co-cultured with autologous CD4+ T cells and proliferation was analysed by [3H]-thymidine incorporation. M = medium control; TT = tetanus toxoid 1 μg/ml; A = native Phleum (Phl) allergen extract; F = Phl formaldehyde-modified allergoid; G = Phl glutaraldehyde modified allergoid. Results are expressed as mean ± standard error of the mean (s.e.m.) from 10 allergic donors. *Statistically significant differences (P < 0·05) between native allergen-and allergoid-pulsed DC.

Further analysis of cytokine production revealed that IL-4 was increased after stimulation of CD4+ T cells with alum-adsorbed glutaraldehyde-modified allergoid-pulsed DC compared to T cells that were stimulated with DC, which were stimulated with unadsorbed glutaraldehyde-allergoids (Fig. 4). However, DC pulsed with alum alone also led to an increase in IL-4 production by T cells compared to untreated or coca buffer-pulsed DC. There were no noteworthy differences between alum-adsorbed native allergen extracts or formaldehyde-allergoids and their corresponding non-adsorbed antigens. IL-13 production of CD4+ T cells was decreased when stimulated by DC pulsed with alum-adsorbed allergens or allergoids compared to DC pulsed with the corresponding unadsorbed antigens. No significant differences could be observed for IL-10 and IFN-γ production (Fig. 4).

Figure 4.

Figure 4

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Cytokine production of CD4+ T cells was mainly unaffected by treatment with alum-adsorbed allergens and allergoids compared to unadsorbed allergens. Alum-adsorbed and unadsorbed allergen-or allergoid-pulsed dendritic cells (DC) were co-cultured with autologous CD4+ T cells. On day 7 T cells were restimulated with freshly generated DC. Interleukin (IL)-4, IL-13, IL-10 and interferon (IFN)-γ production were quantified by enzyme-linked immunosorbent assay (ELISA). M = medium control; TT = tetanus toxoid 1 μg/ml; A = native Phleum (Phl) allergen extract; F = Phl formaldehyde-modified allergoid; G = Phl glutaraldehyde modified allergoid. Results are expressed as mean ± standard error of the mean (s.e.m.) from 10 allergic donors. *Statistically significant differences (P < 0·05) between alum-adsorbed and unadsorbed allergen-pulsed DC.

As already known, glutaraldehyde-modified allergoid-pulsed DC induced lower IL-4, IL-10, IL-13 and IFN-γ production in CD4+ T cells compared with formaldehyde-modified allergoid-and native allergen-pulsed DC [22]. Except for IL-4, similar results were obtained for alum-adsorbed glutaraldehyde-modified allergoid-pulsed DC (Fig. 4).

Alum does not significantly induce IL-1β production by immature DC in vitro

Next, we wondered whether alum affects the Nalp3 inflammasome complex leading to production of proinflammatory cytokines. Therefore, immature DC were stimulated with alum-adsorbed allergens or allergoids or the corresponding non-adsorbed antigens and IL-1β and TNF-α production were analysed by ELISA or CBA 48 h later. LPS-stimulated immature DC or mature DC were used as controls. Compared to mature DC, no or very low amounts of the proinflammatory cytokine IL-1β were detectable in all conditions, and they were not enhanced significantly by alum (data not shown). In further experiments we applied allergen and different alum preparations independently instead of adsorbed alum, but IL-1β production was still low, even after stimulation with LPS. In these LPS-and LPS plus allergen-stimulated DC cultures IL-1β was slightly enhanced by alum (Fig. 5). Similar to IL-1β, allergens or allergoids alone or in combination with alum did not induce the proinflammatory cytokine TNF-α, while it was induced by LPS to the same extent as secreted by mature DC. Addition of alum to LPS plus allergen-stimulated cells also led to a slight increase of this cytokine (Fig. 5). Furthermore, we tested cell lysates of stimulated DC for the presence of intracellular IL-1β, including the biologically inactive precursor. Only one of five donors showed increased levels of IL-1β after stimulation with LPS or LPS plus allergen and alum compared to stimulation with LPS or LPS plus allergen alone (data not shown).

Figure 5.

Figure 5

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Alum does not notably induce interleukin (IL)-1β or tumour necrosis factor (TNF)-α production of immature dendritic cells (DC). Immature DC of allergic donors were stimulated with 10 μg/ml allergen or 100 ng/ml lipopolysaccharide (LPS) ± alum (5 or 10 μg/ml, Alhydrogel 1·3%) and culture supernatants were collected after 48 h. IL-1β and TNF-α production was assessed by quantitative enzyme-linked immunosorbent assay (ELISA). A = native Phleum (Phl) allergen extract; Alum-ad. = alum-adsorbed allergens; mDC = mature DC. Results are expressed as mean ± standard error of the mean (s.e.m.) from five allergic donors.

Besides protein secretion, IL-1β expression was analysed further at the mRNA level. Therefore, immature DC were stimulated with allergen or LPS in the presence or absence of alum and IL-1β mRNA expression was analysed by quantitative reverse transcription–PCR (qRT–PCR) 2 h later, which was chosen as the best time-point in preliminary kinetic experiments. IL-1β mRNA expression was increased compared to the medium control, reaching a maximum change of relative gene expression for LPS and mature DC. Again, a slight but not significant enhancement of IL-1β gene expression could be detected after treatment of immature DC with alum regardless of whether they were stimulated with allergen, LPS, matured or left untreated (data not shown). Effects of the Nalp3 inflammasome, however, should be operational on the protein level.

Alum-adsorbed allergens/allergoids enhance Phl p-specific IgG production in immunized mice

To analyse the influence of alum-adsorbed allergens/allergoids in vivo, BALB/c mice were sensitized six times every second week with alum-adsorbed or unadsorbed Phl p extract or Phl p allergoid, and Phl p-specific IgG was determined weekly. Phl p-specific IgG increased over time, reaching a maximum after the fifth immunization. At every time-point measured, antibody levels were lower in Phl p allergoid-sensitized mice than in mice sensitized with the native Phl p extract. This reduction in Phl p-specific IgG was restored by adsorption of the allergoid to alum (Fig. 6). Alum-adsorbed native Phl p extract-treated mice, however, also produced significantly higher amounts of Phl p-specific IgG compared to mice treated with the unadsorbed allergen extract (Fig. 6).

Figure 6.

Figure 6

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Alum enhances Phleum (Phl) p-specific immunoglobulin (Ig)G in immunized mice. BALB/c mice were immunized with alum-adsorbed or unadsorbed Phl p extract or Phl p allergoid six times every second week and blood samples were taken at the indicated time-points. Shown are the single Phl p-specific IgG values from eight mice after six immunizations (right) or the Phl p-specific IgG kinetic expressed as mean ± standard error of the mean (s.e.m.) from 95% confidence interval.

Discussion

Allergen extracts used for SIT are commonly administered in combination with adjuvants such as alum, which has been reported to have an enhancing effect on the immune response towards the applied antigen as well as to reduce anaphylactic reactions [14,23,25]. However, the exact mechanisms behind the adjuvant effect of alum remain controversial. Therefore, we analysed the effect of alum-adsorbed and unadsorbed allergens and allergoids, modified with formaldehyde or glutaraldehyde, on human immune cells in vitro and on immunized mice in vivo. We could demonstrate that except for an increase in IL-4 production by alum alone, alum-adsorbed allergens/allergoids did not increase T cell or effector cell activation. In contrast, adsorption of allergens/allergoids to alum strongly enhanced IgG production in immunized mice in vivo. Similar observations regarding the dependence of specific IgG antibody responses on the alum concentration have already been shown by us recently [23]. Furthermore, we could confirm our previously published results that T cell proliferation as well as basophil activation were significantly lower when glutaraldehyde-modified allergoids were used [22]. In the study presented here, we show for the first time that the reduction of allergenicity was even more pronounced using alum-adsorbed glutaraldehyde allergoids, as alum-adsorption per se led to a decreased release in most of the donors examined (also with formaldehyde allergoids or native allergens concerning the leukotriene release). The reduced Phl p-specific IgG levels observed after immunization with Phl p allergoid could be restored by the addition of alum, which also enhanced IgG production induced by formaldehyde allergoids or native allergens. Grass allergens, especially group 5, are strongly immunogenic compared to birch allergens [24], and this may explain why differences between allergens and allergoids are less pronounced in the current study. However, all observations point to less immunogenicity of the allergoid preparations, suggesting that statistically significant differences would be obtained for the in-vivo experiments with additional mice in each group (the difference between allergen/allergoid P = 0·08, allergen + alum/allergoid + alum P = 0·08).

The reduced allergenicity of alum-adsorbed glutaraldehyde-modified allergoids was not due to a general toxic effect, as the addition of the highest alum concentration to anti-IgE receptor-stimulated basophils did not influence the release induced by this positive control. However, at the highest concentration of alum-adsorbed antigens used for DC stimulation we observed more dead cells compared to DC stimulated with unadsorbed antigens, which could be confirmed by an apoptosis assay. This finding is consistent with other studies in which a toxic effect of alum at higher concentrations was also reported, related probably to an enhanced cell activation [26,27]. Therefore, dead cells were excluded by trypan blue staining for all co-culture experiments.

To assess the T cell stimulatory capacity of alum-adsorbed allergens and allergoids compared to the corresponding non-adsorbed antigens, we co-cultured allergen-or allergoid-pulsed DC with autologous CD4+ T cells and determined T cell proliferation and cytokine production. Regarding T cell proliferative responses and IL-2 production, we could show that there were no significant differences between alum-adsorbed and unadsorbed allergens and allergoids. Glutaraldehyde-modified alum-adsorbed and unadsorbed allergoids led to a significantly decreased T cell proliferation and IL-2 production compared to the corresponding alum-adsorbed and unadsorbed native allergen extracts and formaldehyde-modified allergoids. This might be due to a more extensive aggregation and modification by glutaraldehyde treatment of our extracts, which was demonstrated previously by our group for unadsorbed allergens and allergoids [22]. In the study presented here, alum was not able to overcome the reduced T cell stimulatory capacity of glutaraldehyde-modified allergoids. Thus, alum had no enhancing effect on T cell proliferation in vitro. However, IL-4 production was increased after stimulation of CD4+ T cells with alum-adsorbed glutaraldehyde allergoid-pulsed DC compared to the correspondent unadsorbed allergoid, while IL-13, IL-10 and IFN-γ production were slightly reduced or unaffected by addition of alum. In line with our results, Ulanova et al. also reported enhanced production of IL-4 together with increased expression of antigen-presenting and co-stimulatory molecules as well as of proinflammatory cytokines (IL-1α, IL-1β, IL-6 and TNF-α) by aluminium hydroxide-treated human peripheral blood monocytes, while production of IL-2, IL-5, IL-10, IFN-γ, TGF-β or granulocyte–macrophage colony-stimulating factor (GM-CSF) was not changed [28]. In addition, in neutralization experiments, they could show that the alum-induced increase in cell surface expression of MHC class II was dependent upon IL-4, and that many cells in the cultures containing aluminum hydroxide acquired similar dendritic morphology, as described for cultured monocyte-derived DC [28,29]. In contrast to IL-4, IL-13 fails to induce Th2 differentiation due to the lack of functional IL-13 receptors on T cells [30], which could also explain why IL-13 is not involved in alum-induced up-regulation of MHC class II.

Activation of human macrophages and of mouse DC by alum has been described further by several groups, including our own observations, that alum led to a strong induction of the DC maturation marker CD83 and to a slight increase of the proinflammatory cytokine IL-1β in immature human DC [23,26,27]. This capacity of alum to stimulate antigen-presenting cells may explain its potent adjuvant effect in vivo, especially for the induction of primary IL-4-driven Th2 immune responses in naive mice [31]. In humans, however, the allergic immune response is already established. Importantly, alum was shown here to down-regulate Th2 cytokine responses by allergen-stimulated PBMC at least in vitro [32], which may be one explanation why alum has been used successfully as adjuvant in the treatment of allergic diseases for many years.

Recently, the Nalp3 inflammasome has been linked to the adjuvant effect of alum, suggesting a stimulatory effect of alum on the release of proinflammatory cytokines such as IL-1β, IL-18 or IL-33 [18,33]. In our study, we investigated the production of IL-1β mRNA (which should not be affected by activation of the Nalp3 inflammasome) and protein by immature DC after stimulation with allergen or LPS in the presence or absence of alum. Both IL-1β mRNA, as well as IL-1β protein expression, were increased slightly but not significantly after treatment of immature DC with alum. Whether this slight but consistent enhancement (at least on the protein level) is due to activation of the Nalp3 inflammasome or due to Toll-like receptor (TLR) signalling is currently under investigation. Preliminary blocking experiments with caspase or TLR-4 inhibitors indicate that both pathways seem to be involved (data not shown). In vivo, however, alum-induced Th2 responses are strongly reduced in Nalp3-knock-out mice but not in mice deficient for the TLR adaptor protein myeloid differentiation primary response gene 88 (MyD88) [17]. Other studies with Nalp3-deficient mice have shown that the adjuvant effect of alum occurred independently from the Nalp3 inflammasome [19–21]. Ovalbumin (OVA)-induced allergic lung inflammation could be inhibited even in the absence of adjuvant [34]. Thus, the relevance of the Nalp3 inflammasome concerning the immunostimulatory effects of alum remains controversial, and needs to be investigated further.

Taken together, our results have demonstrated that alum had only a slight but not significant adjuvant effect in vitro, while it possessed a strong adjuvant activity in allergen-immunized mice in vivo. Due probably to its superior IgG-inducing capacity (immunogenicity), together with the reduced IgE-mediated activation of effector cells, alum is the most common adjuvant for allergen immunotherapy in humans. To conclude, the function of intact allergens as well as allergoids depends upon the balance between allergenicity and immunogenicity, and this balance is shifted by alum.

Acknowledgments

This work was supported by a grant from ALK-Abelló, Hørsholm, Denmark.

Disclosures

L. L., H. H., G. L. and P. A. W are employed by ALK-Abelló A/S. The rest of the authors declare no financial or commercial conflict of interest.

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